cDNA synthesis for BCR-ABL1 detection at the MMR level: the importance of using the appropriate kit

Chi, Jianxiang; Pierides, Chryso; Mitsidou, Andrie; Miltiadou, Andri; Gerasimou, Petroula; Costeas, Paul

doi:10.1186/s12575-015-0014-x

Cited by 4 publications

(3 citation statements)

References 29 publications

(31 reference statements)

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“…[11][12][13] Oncogenic products of alternative splicing, such as EGFR variant III (EGFRvIII) or MET exon 14 skipping (METDex14), are detected by using RT-PCR-based assays, [14][15][16][17] or inferred from DNA-level variants. 15,18 The performance characteristics of these assays are often less than ideal for clinical applications, [9][10][11][12][13][14][15][16][17][18][19][20][21][22] and single-gene assays can be challenging to multiplex, often requiring additional tissue, time, expense, or some combination thereof. As the number of actionable targets grows, the demand for rapid answers from limited specimens also increases.…”

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confidence: 99%

Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts and Oncogenic Isoforms

Sussman

Oran

Paolillo

et al. 2019

Archives of Pathology &Amp; Laboratory Medicine

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Context.— Next-generation sequencing is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for patients with cancer. Oncogenic fusion transcripts are among the various classifications of genetic abnormalities present in tumors and are typically detected clinically with fluorescence in situ hybridization (FISH). However, FISH probes only exist for a limited number of targets, do not provide any information about fusion partners, cannot be multiplex, and have been shown to be limited in specificity for common targets such as ALK. Objective.— To validate an anchored multiplex polymerase chain reaction–based panel for the detection of fusion transcripts in a university hospital–based clinical molecular diagnostics laboratory. Design.— We used 109 unique clinical specimens to validate a custom panel targeting 104 exon boundaries from 17 genes involved in fusions in solid tumors. The panel can accept as little as 100 ng of total nucleic acid from PreservCyt-fixed tissue, and formalin-fixed, paraffin-embedded specimens with as little as 10% tumor nuclei. Results.— Using FISH as the gold standard, this assay has a sensitivity of 88.46% and a specificity of 95.83% for the detection of fusion transcripts involving ALK, RET, and ROS1 in lung adenocarcinomas. Using a validated next-generation sequencing assay as the orthogonal gold standard for the detection of EGFR variant III (EGFRvIII) in glioblastomas, the assay is 92.31% sensitive and 100% specific. Conclusions.— This multiplexed assay is tumor and fusion partner agnostic and will provide clinical utility in therapy selection for patients with solid tumors.

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confidence: 99%

Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts and Oncogenic Isoforms

Sussman

Oran

Paolillo

et al. 2019

Archives of Pathology &Amp; Laboratory Medicine

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“…Purified RNA was reversely transcribed into cDNA with the M-MLV First Strand kit (Invitrogen) according to the manufacturer’s instructions. 15 RT-PCR reactions were carried out using the SYBR Premix Ex Tag II (Takara Bio Inc.) on an ABI PRISM 7500 Sequence Detection system (Applied Biosystems, Foster City, CA, USA). 16 Briefly, after an initial denaturation step at 95°C for 30 s, amplifications were conducted with 40 cycles at a melting temperature of 95°C for 5 s, and an annealing temperature of 60°C for 34 s. Human GAPDH was used as the housekeeping gene to normalize the expression level of target gene.…”

Section: Methodsmentioning

confidence: 99%

LGR6 promotes the progression of gastric cancer through PI3K/AKT/mTOR pathway

Chen

et al. 2018

OTT

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BackgroundIn the present study, we aimed to investigate the role of LGR6 in the progression of gastric cancer (GC) and explore the intrinsic molecular mechanisms.Materials and methodsThe lentiviral LGR6 shRNA (sh-LGR6) and lentiviral expression vector of LGR6 gene (OE-LGR6) were used to regulate the LGR6 expression. Furthermore, we performed in vitro experiments to observe whether PI3K/AKT/mTOR pathway was affected by LGR6 and assess the role of LGR6 in the proliferation, apoptosis, migration, and invasion of GC cells.ResultsOur data showed that phosphorylated AKT and mTOR were downregulated by sh-LGR6 (P<0.05). The expressions of proapoptotic proteins Bax and Caspase-3 were upregulated by sh-LGR6 (P<0.05); the expression of antiapoptotic protein Bcl2 was downregulated by sh-LGR6 (P<0.001). Besides, the functional experiments proved that sh-LGR6 could promote the apoptosis of GC cells and inhibit the proliferation, invasion, and migration of GC cells (P<0.001). Compared with sh-LGR6, OE-LGR6 led to the opposite results.ConclusionLGR6 is an antiapoptosis protein which controls the progression of GC through PI3K/AKT/mTOR pathway. More in vivo experiments and clinical trials are necessary to confirm the possibility of LGR6 in tumor therapy.

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“…CML is divided into three phases—chronic phase, accelerated phase, and blast crisis [ 3 ]. Although found in all age groups, CML more often occurs in middle aged and elderly people with a median age of 67 years [ 4 ]. CML accounts for ~20% of all leukemia cases in adults in western population [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

Hsp90 Inhibitors for the Treatment of Chronic Myeloid Leukemia

Khajapeer

Baskaran

2015

Leukemia Research and Treatment

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Chronic myeloid leukemia (CML) is a hematological malignancy that arises due to reciprocal translocation of 3′ sequences from c-Abelson (ABL) protooncogene of chromosome 9 with 5′ sequence of truncated break point cluster region (BCR) on chromosome 22. BCR-ABL is a functional oncoprotein p210 that exhibits constitutively activated tyrosine kinase causing genomic alteration of hematopoietic stem cells. BCR-ABL specific tyrosine kinase inhibitors (TKIs) successfully block CML progression. However, drug resistance owing to BCR-ABL mutations and overexpression is still an issue. Heat-shock proteins (Hsps) function as molecular chaperones facilitating proper folding of nascent polypeptides. Their increased expression under stressful conditions protects cells by stabilizing unfolded or misfolded peptides. Hsp90 is the major mammalian protein and is required by BCR-ABL for stabilization and maturation. Hsp90 inhibitors destabilize the binding of BCR-ABL protein thus leading to the formation of heteroprotein complex that is eventually degraded by the ubiquitin-proteasome pathway. Results of many novel Hsp90 inhibitors that have entered into various clinical trials are encouraging. The present review targets the current development in the CML treatment by availing Hsp90 specific inhibitors.

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cDNA synthesis for BCR-ABL1 detection at the MMR level: the importance of using the appropriate kit

Cited by 4 publications

References 29 publications

Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts and Oncogenic Isoforms

Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts and Oncogenic Isoforms

LGR6 promotes the progression of gastric cancer through PI3K/AKT/mTOR pathway

Hsp90 Inhibitors for the Treatment of Chronic Myeloid Leukemia

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